Biosensor

ABSTRACT

A biosensor comprises a space part for sucking and housing a sample formed of two upper and lower plates, the two plates being stuck together by an adhesive layer, the space part for sucking and housing the sample being constituted so as to be partially opened in the peripheral part and partially closed by the adhesive layer, and has a working electrode having at least glucose oxidase immobilized thereon and a counter electrode on the same plane of the plate.

This application is a continuation of application Ser. No. 13/181,208,filed Jul. 12, 2011, which is a continuation of Ser. No. 12/068,014,filed Jan. 31, 2008, now U.S. Pat. No. 7,998,336, issued Aug. 16, 2011,which is a continuation of application Ser. No. 11/123,230, filed May 6,2005, now U.S. Pat. No. 7,713,406, issued May 11, 2010, which is acontinuation of application Ser. No. 10/303,084, filed Nov. 25, 2002,now U.S. Pat. No. 6,893,545, issued May 17, 2005, which is acontinuation of Ser. No. 09/664,319, filed Sep. 18, 2000, now U.S. Pat.No. 6,503,381, issued Jan. 7, 2003, which is a divisional of applicationSer. No. 09/484,539, filed Jan. 18, 2000, now U.S. Pat. No. 6,156,173,issued Dec. 5, 2000, which is a continuation of application Ser. No.08/990,997, filed Dec. 15, 1997, now U.S. Pat. No. 6,071,391, issuedJun. 6, 2000, which claims priority to the following Japaneseapplications: JP 09-267812, filed Sep. 12, 1997; JP 09-267814, filedSep. 12, 1997; JP 09-282642, filed Sep. 30, 1997; and JP 09-282643,filed Sep. 30, 1997.

BACKGROUND OF THE INVENTION

This invention relates to a biosensor. More specifically, it relates toa glucose biosensor having glucose oxidase immobilized thereon or abiosensor having an oxidoreductase immobilized thereon.

PRIOR ART

In a conventional biosensor having glucose oxidase immobilized on aworking electrode, a counter electrode or the counter electrode and areference electrodes, in addition to the working electrode, are arrangedon the same plane of a flat base. In a glucose biosensor having theelectrode arrangement as described above, two method are adapted tobring a measuring sample into contact with the working electrode.

The first method, which comprises dropping the measuring sample directlyonto the working electrode, has a problem in that much labor and timeare required from the sampling to the dropping. The second methodinvolves the use of a structure formed of a spacer having a groovearranged on an electrode base and a cover having an air hole furtherarranged thereon to provide a sample suction opening part, a cylindricalpart for sucking and housing the sample, and an air vent hole part. Thismethod has an advantage that it does not take much labor and time sincethe measuring sample is directly guided onto the working electrode, buthas a disadvantage of requiring a complicated process in elementmanufacture such as the setting of the sample suction opening part andthe air vent hole part on both ends of the cylindrical part.

SUMMARY OF THE INVENTION

One object of this invention is to provide a glucose biosensor havingglucose oxidase immobilized thereon.

The other object of this invention is to provide a biosensor easy tomanufacture and measure, and thus suitable also as a disposable glucosebiosensor.

The glucose biosensor of this invention has a structure requiring no airvent hole part.

In the glucose biosensor having glucose oxidase immobilized on anelectrode, the upper and lower parts of a space part for sucking andhousing a sample are formed of two upper and lower plates, the twoplates are mutually stuck by an adhesive layer, the space part forsucking and housing the sample is constituted so as to be partiallyopened in the periphery and partially closed by the adhesive layer, andan electrode structure having at least glucose oxidase immobilizedthereon is provided on the plate, whereby the manufacture andmeasurement are facilitated. When the upper and lower plates aretapered, a target sample can be precisely caught, and the roundness ofthe tip further provides an operational advantage such that the affectedpart is not damaged, for example, in blood sampling on a finger. Thisbiosensor is thus suitable as a disposable glucose biosensor.

The two plates constituting the space part are stuck together only by anadhesive layer or by a spacer with adhesive on both sides. The twoplates are constituted as follows. In the first embodiment, theelectrode structure is formed within the same plane on the same base,and this base is stuck to the other plate (cover) through the adhesivelayer or the like. In the second embodiment, one electrode is formed onone base, and this base is stuck to the other base having one electrodeor two electrodes formed thereon so as to have a facing structure inwhich the electrodes are mutually opposed on the inside. In both cases,the upper and lower parts of the space part for sucking and housing thesample are formed of two plates, the two plates are mutually stuck bythe adhesive layer or the like, the space part for sucking and housingthe sample is constituted so as to be partially opened in the peripheralpart and partially closed by a thick part such as the adhesive layer,and the electrode structure having at least glucose oxidase immobilizedthereon is provided on the plate.

The use of the spacer with adhesive on both sides is given herein as anexample to mutually stick the upper and lower plates, but themanufacturing process can be simplified by the use of only the adhesivelayer as described below. In a biosensor in which a working electrodehaving an oxidoreductase immobilized thereon and its counter electrodeare arranged so as to have a facing structure, for example, each basehaving each electrode on the inside is adhered together through theadhesive layer, whereby an inexpensive manufacturing method can berealized.

The biosensor requires a connector having a special structure since leadparts for ensuring electric continuity are mutually opposed in the innerpart.

Biosensors which do not require such a connector are described below.

(1) A biosensor which comprises a working electrode and a counterelectrode formed on the inside of a longer lower base and a shorterupper base through an adhesive layer or a spacer, respectively, and alead part for each electrode formed in such a manner that the end partis situated in a position on the lower base never superposed on theupper base, the electrode on the upper base being conducted to its leadpart through an adhesive layer or a spacer.

(2) A biosensor which comprises a working electrode and a counterelectrode formed on the inside of a longer upper base and a shorterlower base through an adhesive layer or a spacer, respectively, and alead part for each electrode formed on the surface side of the upperbase, the electrode provided on the upper base being conducted to itslead part through the base, and the electrode provided on the lower basebeing conducted to its lead part provided on the upper base through anadhesive layer or a spacer.

This invention also provides a biosensor device enhanced in reliabilityof the device, improved in a series of operability up to measurementend, and advantageous in cost by avoiding the operation by the wrongrecognition in insertion of a foreign matter other than the sensor.

Such a device comprises an element reaction sensor member to be insertedto the connector part of a device body in such a manner as to beattachable and detachable, the element reaction sensor member havingeach output terminal of a working electrode and a counter electrodeelectrically connected to the connector part-side input terminals, andan element reaction part formed at least on the working electrode. Theelement reaction sensor member further has a sensor insertion judgingelectrode, and the connector part of the device body also has two inputterminals with which the sensor insertion judging electrode outputterminal makes contact, so that the system of the device body is startedby the contact with the two input terminals to judge the sensorinsertion by a control part.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a, 1 b, 1 c are plane views of element components used for themanufacture of a glucose biosensor according to this invention.

FIG. 2 is a plane view of the assembled glucose biosensor.

FIG. 3 is a side view of the assembled glucose biosensor.

FIGS. 4 a, 4 b each are plane views of element components used for themanufacture of a preferable glucose biosensor according to thisinvention.

FIG. 5 is a calibration curve graph showing the relation betweenconcentration of glucose aqueous solution and output.

FIG. 6 is a perspective view of another embodiment of the biosensoraccording to this invention.

FIG. 7 is a plane view of a base having a working electrode providedthereon.

FIG. 8 is a plane view of a base having an insulating layer providedthereon.

FIG. 9 is a plane view of a base having a protruded tip part.

FIG. 10 a is a perspective view of one embodiment of the biosensoraccording to this invention.

FIG. 10 b is a disassembled state view of each component in theembodiment of FIG. 10 a.

FIG. 11 is a perspective view of another embodiment of the biosensoraccording to this invention.

FIG. 12 is a disassembled state view of each component in the embodimentof FIG. 11.

FIG. 13 is a perspective view of the other embodiment of the biosensoraccording to this invention.

FIG. 14 is a disassembled state view of each component in the embodimentof FIG. 13.

FIG. 15 is a system constitution view showing the biosensor device ofthe other embodiment according to this invention.

EMBODIMENTS (A) Embodiments of FIG. 1 a to FIG. 5

FIG. 1 a shows an element having a working electrode 2 and a referenceelectrode lead 3 formed on a base 1, and FIG. 1 b shows an elementhaving a counter electrode 4 formed on a base 1′, and FIG. 1 c shows anelement component formed of a spacer with adhesive on both sides (about100-500 μm in thickness) 5(c). FIG. 2 shows a plane view of an elementassembled from these components. FIG. 3 shows a side view thereof. Thepart denoted at 8 in FIGS. 2, 3 is a space part for sucking and housinga sample. The space part 8 is partially opened in the peripheral part,and partially closed by the spacer with adhesive on both sides.

As the base, plastics such as polyethylene terephthalate (PET) andpolyvinyl chloride, glass, ceramics, paper, biodegradable material (forexample, microorganism producing polyester or the like) are used. Theworking electrode, the counter electrode and the reference electrodelead are formed from platinum, gold, carbon, palladium or the like bymeans of screen printing, vapor deposition, sputtering, blocking or thelike, and a reference electrode 6 is formed by forming a silverelectrode on the reference electrode lead once by screen printing, vapordeposition, sputtering or the like followed by constant currentelectrolysis or dipping in a ferric chloride aqueous solution, or byapplying and laminating silver chloride by screen printing. Although thereference electrode may be set on either of the working electrode-sidebase and the counter electrode-side base, it is preferably set on theworking electrode-side base. A two-electrode structure having noreference electrode is also constituted in the same manner.

Glucose oxidase, which is generally immobilized on a working electrode,may be immobilized on the working electrode periphery, the counterelectrode, or the periphery thereof since it is dissolved to an aqueoussolution which is a measuring sample, and reacted on the workingelectrode.

In the immobilization of glucose oxidase, preferably, onto the workingelectrode, it is formed not only as glucose oxidase single body but alsoas a mixture layer to which at least one of electron acceptor (mediator)and albumin is added as listed below.

(1) Glucose oxidase layer

(2) Glucose oxidase-electron acceptor mixture layer

(3) Glucose oxidase-albumin mixture layer

(4) Glucose oxidase-electron acceptor-albumin mixture layer

The glucose oxidase layer (1) is formed by dissolving about 1-50 mg,preferably about 5-30 mg of glucose oxidase (GOD), for example, in caseof 165800 unit/g GOD, to 1 ml of distilled water or buffer solution,dropping about 0.5-10 μl, preferably about 1-3 μl of the solution (GODsolution) by means of dropping by dispenser or spin coating followed bydrying at room temperature to form a layer about 1-200 μm, preferably,about 50-150 μm in thickness.

In the mixture layers (2)-(4), the same method is employed for theformation, except using a GOD aqueous solution having each of thefollowing components added thereto.

Mixture layer (2): Potassium ferricyanide, parabenzoquinone or the likeis used as the electron acceptor, and a solution to which about 1-100mg, preferably about 30-60 mg in potassium ferricyanide, or about 1-200mg, preferably about 50-150 mg in case of parabenzoquinone is furtheradded is used.

Mixture layer (3): A solution to which about 1-100 mg, preferably about5-30 mg of bovine serum albumin is added is used.

Mixture layer (4): A solution to which the electron acceptor of thequantity used for the formation of the mixture layer (2) and the bovineserum albumin of the quantity used for the formation of the mixturelayer (3) are further added is used.

The added electron acceptor works as described below, and the additionof albumin or the use of buffer solution provides a measurement resultwith less dispersion to pH change of the measuring solution (glucoseaqueous solution).

It is known to indirectly determine glucose concentration by oxidizingglucose under the presence of enzyme by the action of GOD to generategluconolactone, oxidizing the H₂O₂ generated then on the workingelectrode, and measuring the oxidizing current value at that time. Sincethe rate of oxidizing reaction is limited by the dissolved oxygenconcentration in a liquid concentrate sample in which a measuringsolution is not diluted with water, however, the linear calibrationrange is only shown up to about 100 mg/dl of glucose concentration.

Therefore, instead of the oxygen limited in concentration in solution,the electron acceptor is used together with GOD. When the mediator ispotassium ferricyanide K₃Fe(CN)₆, the reaction advances as follows.Glucose+2Fe(CN)₆+H₂O→gluconic acid+2H⁺+2Fe(CN)₆The ferrocyan ion generated then is oxidized by the working electrode togenerate oxidizing current.2Fe(CN)₆→2Fe(CN)₆+2e ⁻

When parabenzoquinone is used instead of potassium ferricyanide asmediator, hydroquinone is generated by the reaction of glucose withparabenzoquinone under the presence of GOD, the generated hydroquinoneis oxidized by the working electrode to generate oxidizing current, andits value is measured.Hydroquinone→parabenzoquinone+2H⁺+2e ⁻

On the other hand, although the counter electrode is usable withoutparticularly immobilizing anything thereon, a layer consisting of atleast one of albumin and electron acceptor may be formed thereon. Inthis case, the inclination of concentration apt to be caused indissolution or dispersion of the mixture layer by the sample solution,which is observed when the mixture layer is provided only on the workingelectrode, is advantageously eliminated, and the measuring precision isalso improved.

In order to smooth the contact of the measuring sample solution with theimmobilized GOD, means such as application of a surface active agentsuch as lecithin, Triton X-100 (Commercial name) or the like onto theworking electrode, the counter electrode, the working electrodeperiphery, the counter electrode periphery, the working electrode andits periphery, or the working electrode and its periphery, or nipping ofan impregnation accelerator such as nonwoven fabric or filter paper byutilizing the clearance of the opening part around the space part may bealso applied.

A low molecule such as sucrose can be also mixed to the above mixture asa moisture retaining agent, and a cross-linking agent such asglutaraldehyde can be further mixed to the above mixture or bonded ontothe electrode to stabilize the immobilization.

The measurement of glucose concentration is performed by bringing about0.5-10 μl of a glucose aqueous solution having a prescribedconcentration into contact with the thus-manufactured glucose biosensorto suck it, applying a voltage of about 0.4-1.2V, preferably about0.6-1.0V thereto after the reaction for about 1-120 seconds, andmeasuring the current value, for example, after 20 seconds from theapplication. A potentiogalvanostat and a function generator are used forthe measurement.

When the glucose biosensor is brought into contact with the glucoseaqueous solution, one end side of each base 1 (1′) is formed into atapered part 8 (8′) as shown in FIG. 4, and the tip part 9 (9′) of theworking electrode 2 (or the counter electrode 4) is provided on thetapered part, preferably on the tip thereof. Namely, FIG. 4 showselement components formed of (a) the base 1 (or 1′) having the workingelectrode 2 (or the counter electrode 4) formed thereon, and (b) thespacer with adhesive on both sides 5, and the bases 1, 1′ can beintegrated together through the spacer 5 with the working electrode 2side and the counter electrode 4 side of the bases 1, 1′ inside. Sincethe separated electrodes are provided on the base tapered parts in sucha sensor, the measuring solution can be directly collected even if it isa trace amount, and the quick contact with the working electrode is thusa great convenience. The side contacting the sample of the base istapered, whereby the intended sample can be precisely caught. Theroundness of the tip also provides an advantage that the affected partis never damaged, for example, in blood sampling on a finger.

The glucose biosensor having glucose oxidase immobilized thereon isconstituted as described above, whereby the manufacture and measurementcan be facilitated, such a glucose biosensor can be thus used as adisposable biosensor for a liquid concentrate sample as solution samplefor domestic medical examination (self-care), particularlyself-management of diabetes, and prevention and early detection ofdiabetes by measurement of blood sugar or urine sugar, and a wide usesuch as use for glucose management in food manufacturing process can beexpected.

Example A1

A counter electrode, a working electrode, and a reference electrode leadeach of which was made of carbon were formed in a film thickness of 5 μmon a polyethylene terephthalate film (0.25 mm in thickness) by screenprinting as the embodiment shown in FIGS. 1-3. A silver paste wasprinted on the reference electrode lead in a thickness of 5 μm by screenprinting followed by baking to form a silver electrode. The silverelectrode part was dipped in 0.1M HCl, and silver chloride was formed onthe surface by performing a constant current electrolysis for 20 minutesat a current density of 0.6 mA/cm.sup.2 to form a silver/silver chloridereference electrode. For this constant current electrolysis, apotentiogalvanostat (manufactured by Hokuto Denko HA501) was used.

Onto the working electrode within each electrode having such astructure, a mixture consisting of 10 mg of glucose oxidase (165800unit) and 48 mg of potassium ferricyanide dissolved in 1 ml ofphosphoric acid buffer solution (pH 7.0) was dropped followed by dryingunder room temperature condition to manufacture two kinds of glucosebiosensors A (using the reference electrode) and B (using no referenceelectrode).

To the manufactured glucose biosensors, 5 μl of a glucose aqueoussolution having a prescribed concentration was introduced from a sampleinlet part to advance the reaction for Seconds, a voltage of 0.6V isthen applied onto the working electrode, and the current value after 20seconds from the application was measured. A potentiogalvanostat (HA501) and a function generator (manufactured by Hokuto Denko HB-104) wereused for the measurement.

The measurement result (output) is shown in the following table and thegraph of FIG. 5.

TABLE Glucose concentration(mg/dl) A B 0 0.5 μA   0 μA 50 1 μA 1 μA 1002 μA 2 μA 250 5 μA 4 μA 500 9 μA 8 μA 800 14 μA  13 μA  1000 18 μA  16μA 

This result shows that linear calibration property within the range of0-1000 mg/dl of glucose concentration can be provided. Each sensor wasdisposed every measurement of one sample. The fluctuation coefficientshowing reproducibility (n=10) of each sensor at the glucoseconcentration of 100 mg/dl was 3.6% in the sensor A, and 3.5% in thesensor B.

(B) Embodiments of FIG. 6 to FIG. 9

FIG. 6 shows a perspective view of another embodiment of the biosensoraccording to this invention, and FIG. 7 shows a plane view of a basehaving a working electrode provided thereon. The part denoted at 11 inFIG. 6 is the space part for sucking and housing a sample.

The base 1 has the working electrode 2, the base 3 has its counterelectrode 4, and an oxidoreductase-electron transmitter mixture layer 5is formed on the working electrode 2 on the base tapered part 8 side. Anadhesive layer 7 is formed in the part excluding the mixture layer andthe lead part 6 of the working electrode 2.

The electrode is preferably used after polished with a nonwoven fabric.

Examples of the oxidoreductase to be immobilized include glucoseoxidase, oxidase lactate, alcohol oxidase, pyruvate oxidase, glucosedehydrogenase, alcohol dehydrogenase, pyruvate dehydrogenase, antibody,and the like, and the concentration of an organic material such asglucose, lactic acid, alcohol, pyruvic acid, antigen or the like can bemeasured by them. The measurement of glucose concentration by glucoseoxidase most generally used is illustrated below through an example ofapplication dying method (adsorption method). In addition to theapplication drying method, conjugation bonding, ion bonding, crosslinking and the like are employed as the immobilizing method of glucoseoxidase.

The base having the working electrode provided thereon and the basehaving the counter electrode provided thereon are generally adheredtogether by use of a double-sided adhesive tape such as double-sidedadhesive nonwoven fabric. The formed adhesive layer must have athickness capable of keeping such a space that the working electrodedoes not make contact with the counter electrode, and it is set to about100-500 μm (about 0.1-0.5 mm), preferably about 150-350 μm (about0.15-0.35 mm).

Instead of the double-sided adhesive tape, an adhesive formed of acrylicresin can be applied to a prescribed position on one or both of thebases by screen printing to adhere both the bases together in the statekeeping the above space. Further, an insulating film 9 formed ofthermosetting polyester resin may be also provided under the adhesivelayer 7 with a length larger than its length in a thickness of about5-25 μm (Referred to FIG. 8).

On end side 8 of the base having the working electrode or counterelectrode is tapered into a pointed form as shown in FIG. 6, so that themeasuring sample can be directly collected even if it is a trace amount,and the contact with the electrode can be thus quickly performed. Theone end side of the base having the electrode provided thereon may bemade also into a protruding form 10 instead of the tapered form.

The biosensor according to this invention in which the working electrodehaving the oxidoreductase immobilized thereon and its counter electrodeare arranged so as to have a facing structure by adhering the baseshaving these electrodes provided on the inside together through theadhesive layer is easy to manufacture, and the manufacturing cost can bealso reduced.

Example B1

Two polyethylene terephthalate bases tapered in one-side ends wereprepared, carbon-made electrodes were formed on the respective bases ina thickness of 10 μm by screen printing. Onto one carbon-made electrode,1.5 μl of a mixture (dopant) consisting of 10 mg of glucose oxidase(165800 unit) and 48 mg of potassium ferricyanide dissolved in 1 ml ofwater was dropped followed by drying under room temperature condition toform a glucose oxidase-potassium ferricyanide mixture layer (about 100μm in thickness) as working electrode.

A base A having the thus-obtained mixture layer formed working electrodeand a base B having its counter electrode were used, and they were stucktogether with a double-sided adhesive tape (Product manufactured byNitto Denko No. 500; 160 μm in thickness) as the adhesive layer invarious embodiments as described below.

(1) The base A having the mixture layer formed working electrode and thebase B having the counter electrode are stuck together by the adhesivelayer provided on the base A side.

(2) The base A having the mixture layer formed working electrode and thebase B are stuck together by the adhesive layer provided on the base Aside.

(3) The bases A having the mixture layer formed working electrodes aremutually stuck by an adhesive layer (one forms the counter electrode).

(4) An insulating layer (formed by use of thermosetting polyester resinin a thickness of 20 μm by screen printing) is provided between eachelectrode and each base in (1) described above.

(5) Each electrode is polished with nonwoven fabric in (1) describedabove.

To the resulting glucose biosensors, 1 μl of a glucose aqueous solution(pH 5.0) having a concentration of 250 mg/dl was sucked, a voltage of0.9V was applied between the working electrode and the counter electrodeafter it is allowed to stand for 20 seconds, and the current value after10 seconds from the application was measured 10 times. Apotentiogalvanostat (manufactured by Hokuto Denko HA501) and a functiongenerator (manufactured by the same company HB-104) were used for themeasurement. When CV value (ratio of standard deviation to averagevalue) was calculated from the measured values, values of (1) 4.5%, (2)4.3%, (3) 4.1%, (4) 4.4%, (5) 4.0% were obtained, respectively. Thesensors were disposed every sample. When the measurement was performedwith the glucose concentration being variously changed, the linearitywas provided within the range of 0-1000 mg/dl.

Example B2

In (1) of Example B1, the glucose aqueous solution was regulated to pH7.0, and a dopant to which 10 mg of albumin was added was used. The CVvalue was 4.8%.

Example B3

In (1) of Example B1, the glucose aqueous solution was regulated to pH7.0, and the dopant was prepared by use of a 0.1M citric acid buffersolution (pH 5.0) instead of water. The CV value was 4.7%.

Example B4

In (1) of Example B1, the glucose aqueous solution was regulated to pH7.0, and the dopant was prepared by adding 10 mg of albumin and using a0.1M citric acid buffer solution (pH 5.0) instead of water. The CV valuewas 4.6%.

Example B5

In Example B4, a nonionic surface active agent (Product manufactured byUCC, Triton X-100) was added to the dopant in a concentration of 0.5 wt.%. The CV value was 4.5%.

Example B6

In (1) of Example B1, a 0.5 wt. % aqueous solution of surface activeagent (Triton X-100) was applied to the periphery of the workingelectrode followed by drying. The CV value was 4.4%.

With respect to the respective glucose biosensors of Example B2-B6, thelinearity was obtained within the range of 0-1000 mg/dl of glucoseconcentration.

(C) Embodiments of FIG. 10 a and FIG. 10 b

FIG. 10 a shows a perspective view of one embodiment of a biosensoraccording to this invention, and FIG. 10 b shows a disassembled stateview thereof.

The base 1 has both a working electrode 2 and the counter electrode 3,and an oxidoreductase-electron transmitter mixture layer 5 is formed onthe working electrode 2 of the tapered part 4 of the base 1. Both thebase 1 and the base (cover) 6 are adhered together by the adhesive layer7. Denoted at 8 in FIG. 10 a is the space part for sucking and housingthe sample. The mixture layer 5 may be formed so as to cover both theworking electrode 2 and the counter electrode 3.

Example C

Two polyethylene terephthalate bases tapered on one-side ends wereprepared, and two carbon-made electrodes were formed on one base in athickness of 10 μm by screen printing. Onto the one carbon-madeelectrode, 1.5 μl of a mixture (dopant) consisting of 10 mg of glucoseoxidase (165800 unit) and 48 mg of potassium ferricyanide dissolved in 1ml of water was dropped followed by drying under room temperaturecondition to form a glucose oxidase-potassium ferricyanide mixture layer(about 100 μm in thickness) as working electrode. The other electrodeformed on the same base was used as counter electrode.

The thus-obtained base A having the mixture layer formed workingelectrode and the counter electrode and the base B (cover) having noelectrode were used, and they were stuck together with a double-sidedadhesive tape (Product manufactured by Nitto Denko No. 500; 160 μm inthickness) as adhesive layer in various embodiments as described below.

(1) The base A and the base B were stuck together by the adhesive layerprovided on the base A side.

(2) An insulating layer (formed 20 μm in thickness by use ofthermosetting polyester resin by screen printing) was provided betweenthe electrode and the base in the above (1).

To these glucose biosensors, 1 μl of a glucose aqueous solution having aconcentration of 250 mg/dl (pH 5.0) was sucked, a voltage of 0.9V wasapplied between the working electrode and the counter electrode after itwas allowed to stand for 20 seconds, and the current value after 10seconds from the application was measured 10 times. Apotentiogalvanostat (manufactured by Hokuto Denko HA 501) and a functiongenerator (manufactured by the same company HB-104) were used for themeasurement. When the CV value (ratio of standard deviation to averagevalue) was calculated from the measured values, values of (1) 4.3%, (2)4.0% were obtained, respectively. The sensors were disposed everysample. When the measurement was performed with the glucoseconcentration being variously changed, the linearity was obtained withinthe range of 0-1000 mg/dl.

(D) Embodiments of FIGS. 11-14

In a biosensor of the embodiment in which one end side of each base istapered, and the tip parts of the working electrode and the counterelectrode are provided on each tapered part, the separated electrodesare provided on the base tapered parts having a pointed shape.Therefore, it is extremely convenient since a measuring solution such asglucose aqueous solution can be directly collected even when it is atrace amount, and the contact with the working electrode can be thusquickly performed. It also has an operational advantage that theaffected part is hardly damaged in blood sampling on finger.

A biosensor having a tapered part is shown in FIG. 11 (perspective view)and FIG. 12 (disassembled state view of each component). The space forsucking and housing the sample is denoted at 36 in FIG. 11.

Two bases, or a longer lower base 1 and a shorter upper base 2 areprepared, and tapered parts 3, 4 are formed on one-end sides of therespective bases. A working electrode 5 and a counter electrode 6 areprovided on the inside of the bases 1, 2, respectively, so that the tipparts are situated on the tapered parts 3, 4, and these electrodes havea facing structure since the respective bases are adhered through anadhesive layer 7 or a spacer 7′.

The lead parts 8, 9 for the electrodes 5, 6 are formed on the lower base1. At that time, each lead part 8, 9 is formed so that the end part issituated in a position never superposed on the upper base 2 in order tolay the end part of each lead part into open state allowing theconnection with a connector requiring no special structure. Theseelectrodes and their lead parts are preferably polished with a cloth.

An oxidoreductase is immobilized on the working electrode situated onthe tapered part tip, and the oxidoreductase is preferably formed as amixture layer 13 with an electron transmitter (mediator). It ispreferred to provide an oxidoreductase-electron transmitter mixturelayer 14 also on the counter electrode situated on the tapered part tip.Plastic insulating films 15, 16 such as thermosetting polyester areprovided about 0.1-0.3 mm in thickness on the working electrode and thecounter electrode on which the mixture layers can not be provided.

To conduct the electrode 6 on the upper base 2 to the lead part 9 formedon the lower base 1, the adhesive layer 7 or the spacer 7′ has a boredpart (through-hole) 10, and a conductive material 11 is filled thereinby silver paste, carbon paste or soldering.

The adhesion of the base having the working electrode and the basehaving the counter electrode by the adhesive layer is generallyperformed by a double-sided adhesive tape such as double-sided adhesivenonwoven fabric. The formed adhesive layer must have a thickness capableof keeping such a space that the working electrode does not make contactwith the counter electrode, and it is set to about 100-500 μm (about0.1-0.5 mm), preferably about 150-350 μm (about 0.15-0.35 mm).

Instead of the double-sided adhesive tape, an adhesive formed of acrylicresin can be applied to a prescribed position on one or both of thebases by screen printing to adhere both the bases together in the statekeeping the above space. Further, an insulating film 16 formed ofthermosetting polyester resin can be provided under the adhesive layer 7with a length larger than its length in a thickness of about 5-25 μm.

The adhesion of the base having the working electrode and the basehaving the counter electrode by the spacer is performed by adhesives 12,12′ on both sides of the spacer.

In the adhesion of each base by the adhesive layer or spacer, it isoperationally easy to provide a hole 20 on the upper base 2 to fill theconductive material 11 through it. In the adhesion by the adhesivelayer, the size of the hole 10 provided on the double adhesive tape 7,for example, is desirably larger than the area of the hole 20 providedon the upper base 2. In this case, the conductive material 11 such assilver paste turns around and makes contact with the exposed part of thelead part 9 of the lower base 1 to ensure a sufficient continuity there.

When a reference electrode is provided, the reference electrode isformed by forming a silver electrode on a reference electrode lead byscreen printing, vapor deposition, or sputtering followed by constantcurrent electrolysis or dipping in ferrous chloride aqueous solution, byapplying and laminating silver chloride by screen printing, or the like.The reference electrode can be set on either of the workingelectrode-side base and the counter electrode-side base, but it ispreferably set on the working electrode-side base. Actually, theelectrode is formed on the same base so as to draw the lead part to thesurface side through the through-hole in parallel to the otherelectrode. The reference electrode part must not be covered with themixture layer.

FIG. 14 shows a disassembled state view of each component in anotherembodiment having a tapered part. The part denoted at 37 in FIG. 13 isthe space part for sucking and housing a sample.

A working electrode 21 and a counter electrode 22 are formed on theinside of a longer upper base 24 and a shorter lower base 25 through anadhesive layer 23 or a spacer 23′ so as to have a facing structure,respectively, and the lead parts 26, 27 of the electrodes are formed onthe surface side of the upper base 24, respectively, in the same manneras in the embodiment of FIGS. 11-12. The formation of mixture layers 34,35 onto the working electrode 21 and the counter electrode 22 is alsoperformed in the same manner as the embodiment of FIGS. 11-12.

In the embodiment of FIG. 14, the upper base 24 has a bored part(through-hole) 28 so as to conduct the electrode 21 provided on theinside (reverse side) of the upper base 24 to the lead part 26 providedon the surface side, a conductive material 29 is filled therein, and aconductive material 31 is filled in the through-hole 30 of the adhesivelayer 23 or the spacer 23′ in order to conduct the electrode 22 providedon the lower base 25 to the lead part 27 provided on the upper electrode24. Denoted at 32, 33 are insulating films.

As the base, insulating bases such as plastic represented bypolyethylene terephthalate, biodegradable plastics, glass, ceramics,paper are used in film, sheet or plate form. The formation of theworking electrode, the counter electrode, and their lead parts isperformed by means of screen printing using paste of carbon, silver, orgold or foil application using palladium foil onto both sides borderedwith the bored part.

Although the biosensor having the facing structure by interposing theadhesive layer or spacer between the base having the working electrodearranged thereon and the base having the counter electrode arrangedthereon required a connector having a special structure since theelectrode lead parts are mutually opposed in the inner part, the leadparts of the working electrode and the counter electrode are made intoopened end parts, whereby a biosensor requiring no special connector canbe provided.

Such a biosensor is easy to manufacture and measure by setting theworking electrode and the counter electrode so as to have a facingstructure, and can be effectively used, as a disposable biosensor usinga liquid concentrate sample as measuring solution, for domestic medicalexamination (self-care), particularly, self-management of diabetes andprevention and early detection of diabetes by measurement of blood sugaror urine sugar, and a wide use such as use for glucose management infood manufacturing process can be expected.

Example D1

The biosensor of the embodiment shown in FIGS. 11-12 was manufactured asfollows.

Two long and short polyethylene terephthalate films (0.25 mm inthickness) were prepared as a lower base and an upper base,respectively, and a working electrode, its lead part, and a counterelectrode lead part were formed on the lower base, and a counterelectrode and a part of its lead part on the upper base in a width of1.0 mm and a thickness of 10 μm by screen printing using carbon paste,respectively. Further, prescribed insulating films were provided on theworking electrode and the counter electrode by screen printing usingthermosetting polyester.

On the working electrode of the longer polyethylene terephthalate filmhaving the thus-formed working electrode, 1.5 μl of a mixture (dopesolution) consisting of 10 mg of glucose oxidase (165800 unit/g) and 48mg of potassium ferricyanide dissolved in 1 ml of water was droppedfollowed by drying under room temperature condition to form a mixturelayer. The mixture layer was also formed on the counter electrode in thesame manner. Prior to the formation of the mixture layers, the workingelectrode part and the counter electrode part were polished withnonwoven fabric.

The mixture layer formed electrode bases were mutually stuck by use of aspacer with adhesive on both sides (material: polyethyleneterephthalate, thickness: 0.25 mm) to manufacture a glucose biosensorhaving electrodes of the facing structure. Silver was filled in thethrough-hole (diameter: 0.8 mm) of the spacer by applying silver pasteto ensure the continuity between the counter electrode and its leadpart.

To the above glucose biosensor, 1 μl of a glucose aqueous solutionsample (concentration: 250 mg/ml) of pH 5.0 was sucked, a voltage of0.9V was applied between the working electrode and the counter electrodeafter it was allowed to stand for 80 seconds, and the current value(unit: μA) after 10 seconds from the application was measured. Themeasurement was performed five times, and the average value and the CVvalue (ratio of standard deviation to average value) were calculated. Apotentiogalvanostat (manufactured by Hokuto Denko HA501) and a functiongenerator (manufactured by the same company HB-104) were used for themeasurement, and the above glucose biosensor was mounted on this deviceto perform the measurement. The sensor was disposed every samplemeasurement.

24.0 22.5 24.5 23.0 26.0 Average Value: 24.0 CV Value: 5.7%

Example D2

The biosensor of the embodiment shown in FIGS. 13-14 was manufactured asfollows.

Two long and short polyethylene terephthalate films were prepared as anupper base and a lower base, and a working electrode, its lead part, anda counter electrode lead part were formed on the upper base, and acounter electrode and a part of the lead part on the lower base in awidth 1.0 mm and a thickness 10 μm by screen printing using carbonpaste, respectively. A prescribed insulating film was provided in thesame manner as in Example D1. The formation of the mixture layers ontothe working electrode and the counter electrode was also performed inthe same manner as in Example D1.

These mixture layer-formed electrode bases were mutually stuck with aspacer with adhesive on both sides to manufacture a glucose biosensorhaving electrodes of the facing structure. Silver paste was applied toeach through-hole of the upper base and the spacer to fill silver,whereby the continuity of the working electrode and its lead part to thecounter electrode and its lead part was ensured.

The measurement by use of the thus-manufactured glucose biosensor wasperformed in the same manner as in Example D1 to provide the followingresult.

24.0 22.0 24.5 24.5 26.0 Average Value: 24.2 CV Value: 6.0%

Example D3

A double-sided adhesive tape (Product manufactured by Nitto Denko No.500, thickness: 0.16 mm) was used instead of the spacer with adhesive onboth sides in Example D1. The size of the hole bored in the double-sidedadhesive tape was set larger than the size of the hole provided on theupper base.

The measurement by use of the thus-manufactured glucose biosensor wasperformed in the same manner as in Example D1, and the following resultwas obtained.

24.0 24.5 22.5 23.0 24.0 Average Value: 23.7 CV Value: 3.8%

Example D4

The mixture layer was formed only on the working electrode in ExampleD3, and the following result was obtained.

25.9 24.0 23.0 24.5 23.0 Average Value: 24.1 CV Value: 5.0%

In each of Examples D1-D4 described above, linearity was provided withinthe calibration range of 0-1000 mg/dl of glucose aqueous solutionconcentration.

(E) Embodiments of FIG. 15

Bioelectronic studies for applying biodynamics to electronic field havebeen advanced. A biosensor in this bioelectronic field is a deviceutilizing the excellent molecule identifying function possessed byorganism, which is regarded as a promising device capable of quickly andeasily measuring a chemical material. Such a biosensor is applied as atrace sample measuring sensor, and has a wide applicable field such asdisposable use for domestic medical examination (self-care) of measuringblood sugar or urine sugar to self-manage and prevent diabetes, andindustrial use for sampling quality inspection of product on productionline.

As a concrete example of measurement, a material to be measured in acollected aqueous solution sample is dropped to a reaction part, and thereduced material generated by, for example, enzyme reaction is oxidized,whereby the element current value by the oxidation is taken out anddetected. The measured value equivalent to the element current value isdetermined in reference to a data table, and it is outputted anddisplayed.

In such a biosensor, an optical system is used as a judging means forrecognizing whether the sensor is inserted into the device body and laidin measurable state or not to detect the reflected light or transmittedlight changed by the insertion of the sensor.

The biosensor device according to this invention is enhanced inreliability of the device, improved in a series of operability up tomeasurement end by avoiding the operation by the wrong recognition ininsertion of a foreign matter other than the sensor, and alsoadvantageous in cost.

In the biosensor device of this invention, an element reaction sensormember inserted to the connector part of a device body in such a manneras to be attachable and detachable has each output terminal of a workingelectrode and a counter electrode to be electrically connected to acorresponding input terminal on connector part side by insertion, and anelement reaction part is formed on at least the working electrode of theworking electrode and the counter electrode. The element reaction sensormember further has a sensor insertion judging electrode, and theconnector part of the device body has two input terminals with which thesensor insertion judging electrode output terminal makes contact, sothat the system of the device body is started by the contact with thetwo input terminals to judge the insertion of the sensor by a controlpart.

In this case, the control part judges the dropping of a material to bemeasured by a signal for laying the working electrode and the counterelectrode into close circuit by the dropping of the material to theelement reaction part, starts to count the residual time up to a presetmeasurement end, and transmits the count signal to a display partprovided on the device body to display it.

FIG. 15 shows a system constitution view of a biosensor device accordingto the other embodiment of this invention. The system mainly has asensor 1 for taking the current value generated by an element reactionby dropping an aqueous solution sample to be measured, and a device body10 for converting and displaying the taken current value to anequivalent measured value. The sensor 1 is disposable, and easilyattachable and detachable to a connector part 11 provided on the devicebody 10 side.

For a rectangular insulating base 2 for forming the body of the sensor1, ceramics, glass, paper, biodegradable material (for example,microorganism producing polyester) and plastic material such aspolyethylene terephthalate are used. A pair of electrodes 3, 4 fortaking the element current generated by enzyme reaction of, for example,an oxidoreductase are pattern-formed on the base 2. Both the electrodes3, 4 can be defined as the names of the working electrode 3 and thecounter electrode 4. As the electrode material, conductive metals suchas carbon, silver, gold, palladium are used, and they are pattern-formedby screen printing, sticking, vapor deposition, or sputtering.

A mixture layer 5 which is the element reaction part is formed on theworking electrode 3 or on both the working electrode 3 and the counterelectrode 4. The mixture layer 5 can be formed by use of a mixture of anoxidoreductase and an electron transmitter (mediator), for example, amixture of glucose oxidase and potassium ferricyanide. In most ofdisposable glucose biosensors using glucose oxidase which is a typicaloxidoreductase, the liquid concentrate sample of a material to bemeasured is collected for measurement. The method of indirectlydetermining the glucose aqueous solution concentration by the elementcurrent value by oxidation is known, and it comprises generatinggluconolactone simultaneously with reducing ferricyan ion to formferrocyan ion by glucose oxidase effect, and oxidizing the ferrocyan ionon the working electrode 3 to detect and measure the element currentvalue.

The respective end parts (lead parts) of the working electrode 3 and thecounter electrode 4 on the opposite side to the position having themixture layer 5 are formed as a pair of opposed output terminals 6, 7. Asensor insertion judging electrode (hereinafter referred to as sensorinsertion signal terminal) 8 which is the essential member of thisinvention is formed on the base 2 between the output terminals 6, 7.This sensor insertion signal terminal 8 is generally formed of the samematerial as the working electrode 3 and the counter electrode 4 in thesame forming method. An insulating layer 9 is formed on a part of thebase 2 by screen printing by use of thermosetting polyester material soas to lay the terminals 6, 7, 8 into the mutually electrically insulatedstate.

On the other hand, the device body 10 is formed of the following parts.It has a connector part 11 for the part for inserting and electricallyconnecting the disposable sensor 1 in measurement. The connector part 11is formed of four connector pins in total of an input terminal 12 to beconnected to the output terminal 6 of the working electrode 3 on thesensor 1 side, an input terminal 13 connected to the output terminal 7of the counter electrode 4, and two input terminals 14 a, 14 bcorresponding to the sensor insertion signal terminal 8. Of the twoinput terminals 14 a, 14 b provided in conformation to the sensorinsertion signal terminal 8, one input terminal 14 a is connected to aregulator part 15, and the other input terminal 14 b to a power sourcecircuit 16. Thus, the two input terminals 14 a, 14 b are connected inshort-circuit state by the contact with the signal terminal 8 by theinsertion of the sensor from the open circuit state to form a closedcircuit, and the regulator part 15 is connected to the power sourcecircuit 16, whereby a power is supplied to a control part 20 to bedescribed later to start the system. Namely, the sensor insertion signalterminal 8 is provided as the essential member in order to detect andjudge the insertion of the sensor 1 to the device body 10 and start upthe system.

As the part for controlling the system, the device body 10 has a controlpart 20 formed of a CPU (central processing unit) by microcomputer. Thecontrol part 20 is formed of a current-voltage converting circuit 21 forconverting the detected current to voltage value, an amplifying circuit22 for amplifying the converted voltage signal, an arithmetic part 23for arithmetically processing on the basis of the input data signal, anda display part 24 such as LCD (liquid crystal display device) fordigitally displaying the value processed in the arithmetic part 23 asmeasurement data. In the CPU, the entire control is performed on thebasis of signals inputted and outputted through an I/O port from eachpart and each circuit.

The operation and action of the biosensor device described above areillustrated. In measurement, the sensor 1 is inserted to the connectorpart 11 of the device body 10. In this sensor inserting stage, anaqueous solution sample containing a collected material to be measuredis not dropped to the mixture layer 5 of the element reaction part onthe sensor 1 side. Thus, the working electrode 3 and the counterelectrode 4 of the sensor 1 are still in open state.

Both the output terminals 6, 7 of the working electrode 3 and thecounter electrode 4 of the sensor 1 are connected to the correspondinginput terminals 12, 13 on the device body 10 side by the insertion ofthe sensor. The sensor insertion signal terminal 8 is also connected tothe two input terminals 14 a, 14 b in the connector part 11 of thedevice body 10. Then, they are laid into short-circuit state to form aclosed circuit, the regulator part 15 is connected to the power sourcecircuit 16 to supply a power to the control part 20. The control part 20judges the insertion of the sensor 1 by the starting of the system. Avoltage is applied between the working electrode 3 and the counterelectrode 4 by a control signal outputted from the control part 20according to this judgment of sensor insertion.

When a 0.5 wt. % of glucose aqueous solution sample, for example, isdropped onto the mixture layer 5 of the sensor 1, the working electrode3 and the counter electrode 4 are laid in short-circuit state to form aclosed circuit. According to this close signal, the control part 20judges the dropping of the glucose aqueous solution sample, andinterrupts the voltage supply applied between the working electrode 3and the counter electrode 4. Synchronously to this, the control part 20starts to count the residual time to the measurement end with a presettime numerical as starting point, for example, 30 seconds. The countedresidual time is displayed on the display part 24 so as to berecognizable by an operator. When the residual time reaches a set time,the voltage of a preset value for reaction is applied between theworking electrode 3 and the counter electrode 4. By this voltagereapplication, the reduced material generated by the enzyme reaction ofthe glucose aqueous solution sample dropped onto the mixture layer 5 isoxidized on the working electrode 3, and the element current valuegenerated by the oxidation is detected and read.

After the reacting voltage is applied only for a fixed time, thedetected and read element current value is converted into voltage valuein the current-voltage converting circuit 21, and the converted voltagevalue is amplified by the amplifying circuit 22. The calculation resultdata in the arithmetic part 23 in reference to the data tablecorresponding to the amplified voltage is displayed on the display part24.

When the sensor 1 is not removed from the device body 10 after themeasurement result data is displayed for a fixed time, the power sourcecircuit 16 is OFF. Even when the sensor 1 is laid into leaving state,the power source circuit 16 on the device body 10 side can beautomatically OFF after the lapse of, for example, 2 minutes. The powersource circuit 16 can be switched OFF also by removing the sensor 1.

The sensor 1 having the working electrode 3 and the counter electrode 4flat pattern-formed within the same plane on the rectangular body base 2was described above as an example, but this invention is not limited bythis electrode pattern. For example, the working electrode 3 and thecounter electrode 4 are formed on the inside of the opposed bases,whereby a facing structure can be formed. The arranging position of thesensor insertion signal terminal 8 is also optionally selected. Further,the display form and display character style for display of measurablestate, count display of measuring time, concentration display of anintended material in a collected sample, and the like can be alsooptionally set.

According to the biosensor device of this invention, since the insertionof the sensor to the device body can be surely detected by providing theinsertion signal terminal formed of the sensor insertion judgingelectrode on the sensor side, there is no fear of causing a trouble suchas operation by the wrong recognition in insertion of a foreign matterother than the sensor as in the past. Since the sensor insertion isdetected by an electric signal without using a conventional complicatedand expensive optical system as the means for detecting and judging thesensor insertion, this device is suitable for an inexpensive and easydisposable device.

Example E1

When an element reaction sensor member having a working electrode and acounter electrode by screen printing using carbon paste, a sensorinsertion judging electrode by screen printing using silver paste, and athermosetting polyester insulating layer by screen printing formed on apolyethylene terephthalate base, respectively, was inserted to theconnector part of a system body, a power source was automatically ON,and “READY” was displayed on a display part.

When a 500 mg/dl glucose aqueous solution was dropped onto a sensormixture layer (mixture layer of glucose oxidase and potassiumferricyanide), “residual time 30 seconds” was displayed on the bodydisplay part, and the countdown of the residual time was started. When“residual time 10 seconds” was displayed, a voltage of 1.0V was appliedbetween both the electrodes, the reduced material generated by theenzyme reaction was oxidized by the working electrode surface after 10seconds from it, the oxidizing current value generated then was readinto the body circuit part within the system, and converted into voltagefollowed by amplification, and the value according to it was determinedin reference to a data table. As a display value, “500 mg/dl” wasoutputted to the display part. When the sensor was left as in theinserted state, the power source of the body was automatically OFF after2 minutes.

What is claimed is:
 1. A biosensor for determining a concentration ofglucose in a sample, the biosensor comprising: a first substrate and asecond substrate disposed over the first substrate, wherein the firstand second substrates define a sensor comprising a proximal end and adistal end, the distal end being configured for insertion into a sensorreader, wherein the proximal end comprises a narrow portion as comparedto the distal end, and wherein the first substrate comprises a lengthextending from the proximal end to the distal end and the secondsubstrate comprises a length extending from the proximal end to thedistal end, wherein the length of the second substrate is greater thanthe length of the first substrate; a spacer layer disposed between thefirst and second substrates and defining an aperture along a first sideedge of the sensor configured to receive a sample fluid; a workingelectrode disposed on the narrow portion of the proximal end of thefirst substrate and extending to the distal end of the first substrate;a mediator and a glucose dehydrogenase disposed on the workingelectrode; and a counter/reference electrode disposed on the narrowportion of the proximal end of the second substrate and extending to thedistal end of the second substrate.
 2. The biosensor of claim 1, whereinthe spacer layer comprises a length extending from the proximal end tothe distal end, wherein the length of the spacer layer is shorter thanthe length of the first substrate and the length of the secondsubstrate.
 3. The biosensor of claim 1, wherein the first substrate,second substrate and spacer layer define a space for housing a sample,the space for housing a sample being in communication with the apertureand having a volume that is no more than 1.0 μL.
 4. The biosensor ofclaim 3, wherein the space for housing a sample has a volume that is nomore than 0.5 μL.
 5. The biosensor of claim 1, wherein the spacer layerdefines an aperture along a second side edge of the biosensor configuredto receive a sample fluid.
 6. The biosensor of claim 5, wherein theaperture along the first side edge is in communication with the aperturealong the second side edge.
 7. The biosensor of claim 1, wherein theworking electrode is constructed of a palladium material.
 8. Thebiosensor of claim 1, wherein the counter/reference electrode isconstructed of a gold material.
 9. The biosensor of claim 1, wherein themediator is potassium ferricyanide or parabenzoquinone.
 10. A method ofdetermining a concentration of glucose in a subject, the methodcomprising: contacting body fluid of the subject with a biosensor togenerate an electrical signal, the biosensor comprising: a firstsubstrate and a second substrate disposed over the first substrate,wherein the first and second substrates define a sensor comprising aproximal end and a distal end, the distal end being configured forinsertion into a sensor reader, wherein the proximal end comprises anarrow portion as compared to the distal end, and wherein the firstsubstrate comprises a length extending from the proximal end to thedistal end and the second substrate comprises a length extending fromthe proximal end to the distal end, wherein the length of the secondsubstrate is greater than the length of the first substrate; a spacerlayer disposed between the first and second substrates and defining anaperture along a first side edge of the sensor configured to receive asample fluid; a working electrode disposed on the narrow portion of theproximal end of the first substrate and extending to the distal end ofthe first substrate; a mediator and a glucose dehydrogenase disposed onthe working electrode; and a counter/reference electrode disposed on thenarrow portion of the proximal end of the second substrate and extendingto the distal end of the second substrate; and determining theconcentration of glucose in the body fluid using the generatedelectrical signal.
 11. The method of claim 10, wherein determining theconcentration of glucose comprises determining the concentration ofglucose by amperometry using the generated electrical signal.
 12. Themethod of claim 10, wherein the spacer layer comprises a lengthextending from the proximal end to the distal end, wherein the length ofthe spacer layer is shorter than the length of the first substrate andthe length of the second substrate.
 13. The method of claim 10, whereinthe first substrate, second substrate and spacer layer define a spacefor housing a sample, the space for housing a sample being incommunication with the aperture and having a volume that is no more than1.0 μL.
 14. The method of claim 13, wherein the space for housing asample has a volume that is no more than 0.5 μL.
 15. The method of claim10, wherein the spacer layer defines an aperture along a second sideedge of the biosensor configured to receive a sample fluid.
 16. Themethod of claim 15, wherein the aperture along the first side edge is incommunication with the aperture along the second side edge.
 17. Themethod of claim 10, wherein the working electrode is constructed of apalladium material.
 18. The method of claim 10, wherein thecounter/reference electrode is constructed of a gold material.
 19. Themethod of claim 10, wherein the mediator is potassium ferricyanide orparabenzoquinone.
 20. A biosensor device comprising: a device bodyhaving a connector part and a control part, the connector part havinginput terminals; a biosensor comprising: a first substrate and a secondsubstrate disposed over the first substrate, wherein the first andsecond substrates define a sensor comprising a proximal end and a distalend, the distal end being configured for insertion into a sensor reader,wherein the proximal end comprises a narrow portion as compared to thedistal end, and wherein the first substrate comprises a length extendingfrom the proximal end to the distal end and the second substratecomprises a length extending from the proximal end to the distal end,wherein the length of the second substrate is greater than the length ofthe first substrate; a spacer layer disposed between the first andsecond substrates and defining an aperture along a first side edge ofthe sensor configured to receive a sample fluid; a working electrodedisposed on the narrow portion of the proximal end of the firstsubstrate and extending to the distal end of the first substrate; amediator and a glucose dehydrogenase disposed on the working electrode;and a counter/reference electrode disposed on the narrow portion of theproximal end of the second substrate and extending to the distal end ofthe second substrate, wherein the input terminals of the connector partare electrically connected to the working electrode when the biosensoris inserted into the connector part of the device body.
 21. Thebiosensor device of claim 20, wherein the spacer layer comprises alength extending from the proximal end to the distal end, wherein thelength of the spacer layer is shorter than the length of the firstsubstrate and the length of the second substrate.
 22. The biosensordevice of claim 20, wherein the first substrate, second substrate andspacer layer define a space for housing a sample, the space for housinga sample being in communication with the aperture and having a volumethat is no more than 1.0 μL.
 23. The biosensor device of claim 22,wherein the space for housing a sample has a volume that is no more than0.5 μL.
 24. The biosensor device of claim 20, wherein the spacer layerdefines an aperture along a second side edge of the biosensor configuredto receive a sample fluid.
 25. The biosensor device of claim 24, whereinthe aperture along the first side edge is in communication with theaperture along the second side edge.
 26. The biosensor device of claim20, wherein the working electrode is constructed of a palladiummaterial.
 27. The biosensor device of claim 20, wherein thecounter/reference electrode is constructed of a gold material.
 28. Thebiosensor device of claim 20, wherein the mediator is potassiumferricyanide or parabenzoquinone.
 29. A biosensor comprising: aninsulating material having a first surface and a second surface; a firstportion of an electrode disposed on the first surface of the insulatingmaterial; a second portion of the electrode disposed on the secondsurface of the insulating material, a through-hole located in theinsulating material and in contact with the first portion of theelectrode on the first surface of the insulating material and the secondportion of the electrode on the second surface of the insulatingmaterial; wherein the through-hole is filled with a conductive materialthereby providing electrical communication between the first portion ofthe electrode on the first surface of the insulating material and thesecond portion of the electrode on the second surface of the insulatingmaterial.
 30. The biosensor of claim 29, wherein the through-hole isfilled with carbon paste.
 31. The biosensor of claim 29, furthercomprising a second electrode.
 32. The biosensor of claim 29, whereinthe electrode is a counter electrode.
 33. The biosensor of claim 32,wherein the counter electrode comprises platinum, gold, carbon, orpalladium.
 34. The biosensor of claim 29, wherein the electrode is aworking electrode.
 35. The biosensor of claim 34, wherein the workingelectrode comprises platinum, gold, carbon, or palladium.
 36. Thebiosensor of claim 34, wherein the working electrode comprises ananalyte-responsive enzyme selected from the group consisting of glucoseoxidase or glucose dehydrogenase.
 37. The biosensor of claim 29, whereinthe biosensor comprises a proximal end and a distal end, the distal endbeing configured for attachment to a device that measures the analyte,wherein the proximal end comprises a narrow portion as compared to thedistal end.